extraction, isolation and characterization of lectins from

University of Mentouri Brothers Constantine 1

Faculty of Nature and Life Sciences

Dissertation To Get a Diploma of Master in Biochemistry

Option: Molecular Nutrition and Health

Entitled:

:Done by

BENSEGHIR Chaima NAMULODI Phiona

Examination board:

President

Dr. T. NOUADRI

M. C. A. University of MENTOURI Brothers Constantine 1

Supervisor

Prof. Y. NECIB

Pr. University of MENTOURI Brothers Constantine 1

Examiner

Dr. F. MEROUANE

M. C. B. National School of Biotechnology

Academic year

2017-2018

الجمهورية الجزائرية الديمقراطية الشعبية

People’s Democratic Republic of Algeria

وزارة التعليم العالي و البحث العلمي

Ministry of Higher Education and Scientific Research

قسم الكيمياء الحيوية و البيولوجيا الخلوية و الجزيئية

Department of Biochemistry /Molecular and Cellular Biology

جامعة الاخوة منتوري قسنطينة

1

كلية العلوم الطبيعة و الحياة

Extraction, Isolation and Characterization

of Lectins from Moringa oleifera seeds

Acknowledgement

We bless the Almighty God, who in the abundance of His

goodness has given us grace, ability strength, willingness and

patience to do this research.

Our sincere gratitude goes out to our supervisor, Professor

NECIB Y, who despite of his tight schedule has guided us

through this work, for his understanding, his availability, his

patience and his help all along this period of research.

With deep appreciation, we thank our panel of judges, the

President of the jury: Mr. NOUADRI Tahar, (M.C.A University of

Mentouri Brothers, Constantine 01) and the Examiner:

Mr.MEROUANE Fateh, (M.C.B National School of Biotechnology)

who accepted to evaluate and examine our work.

We cannot forget to thank Dr TOUMI S for having guided us all

along in the practical part of our research, thank you for the

time you sacrificed for us.

And to all those that contributed to the completion of this work

through your advice and support, thank you.

THANK YOU

Dedication To my family, both immediate and extended, especially my

mother, Ms Wiiwo J for the unconditional love, inspiration

and relentless support;

To my father, the late Hasunahi S, for being the best dad I

could ever ask for;

To my friends and everyone that guided and supported me

physically and spiritually;

To all my teachers and educators who have inspired and

helped me acquire knowledge;

To the Algerian and Ugandan governments, from whom I

obtained the scholarship;

To my colleague Chaima B with whom I have shared the ups

and downs during this research, and to her family, for being

a family to me, and

To everyone that loves science,

This is dedicated to each one of you!!!

Phiona

Dedication To my family, both immediate and extended, especially my

mother for the unconditional love, inspiration and relentless

support;

To my father for being the best dad I could ever ask for;

To my friends and everyone that guided and supported me

physically and spiritually;

To all my teachers and educators who have inspired and

helped me acquire knowledge;

To my colleague Phiona with whom I have shared the ups

and downs during this research and

To everyone that loves science,

This is dedicated to each one of you!!!

Chaima

Abstract

The purpose of the research was to study the isolation, purification and

characterization of lectins from mature Moringa oleifera seeds. The lectins were

extracted from the seeds with phosphate-buffered saline (PBS) at pH 7.4, and partial

purification was accomplished by ammonium sulfate precipitation and gel filtration

using Sephadex G-50.

Agglutination using glutaraldehyde-treated rabbit erythrocytes and human blood types

A, B, O was carried to determine the presence and specificity of the lectin.

Subsequently, the Moringa oleifera seed lectins were classified as a complete non-

specific lectin since it agglutinated all human blood types and glutaldehyde-treated

rabbit erythrocytes.

The results of carbohydrate specificity showed that the lectin had complex sugar

specificity to fetuin. The lectins were found to be thermally stable, and stable within the

pH range of 3-6. It was also considered as a non metallolectin since its activity was not

significantly affected by EDTA.

Keywords: Lectins, Extraction, Agglutination, Characterization, Moringa oleifera,

Inhibition test.

Résumé

Le but de la recherche était d'étudier l'isolement, la purification et la

caractérisation des lectines des graines mûres de Moringa oleifera. Les lectines ont été

extraites des graines avec une solution saline tamponnée au phosphate (PBS) à pH 7,4 et

la purification a été effectuée par précipitation au sulfate d'ammonium et filtration sur

gel en utilisant Sephadex G-50.

Une teste d’agglutination utilisant des érythrocytes de lapin fixé par glutaraldéhyde et

des groupes sanguins humains A, B, O a été effectué pour déterminer la présence et la

spécificité de la lectine. Les lectines des graines de Moringa oleifera ont été classée

comme des lectines complète non spécifique puisqu'elles agglutinaient tous les types de

sang humain et les érythrocytes de lapin fixé par glutaraldéhyde.

Les résultats de la spécificité glucidique ont montré que les lectines ont une

spécificité pour la fétuine. Les lectines sont révélées thermiquement stable et stable dans

la gamme de pH de 3-6. Elles ont également été considérées comme des non metallo

lectines car l’addition d’EDTA n’a pas un effet considérable sur l’activité.

Mots clés: Lectines, Extraction, Agglutination, Characterisation, Moringa oleifera,

Le test d’inhibition.

ملخص

كتين المستخلص من بذوريالهدف من هذا البحث هو دراسة عزل و تنقية و خصائص الل

.الناضجة

و ، 7,4الحموضة كتين من البذور بنقعه في محلول ملحي ) فوسفات مخزنة مالحة ( في درجة يتم استخراج الل

هلامتمت تنقيته بعد ترسيب كبريتات الامونيوم باستخدام الترشيح و

ب تنفيذ اختبار التراص باستعمال كريات الدم الحمراء للارنب المعالجلتحديد وجود اللكتين و معرفة خصائصه تم

و انواع الدم البشري

صنف اللكتين المستخلص من البذور من بين اللكتينات الكاملة نظرا لالتصاقه بجميع انواع الدم البشري و خلايا الدم

نب المعالج ب الحمراء للار

ان خصائصه و ، اظهرت نتائج نوعية الكربوهيدرات ان اللقاح لديه خصوصية اتجاه السكر المعقد

مركب كامل لعدم كما تم اعتباره .6الى 3في استقراره حراريا و استقراره ضمن نطاق درجة الحموضة من تتمثل

تاثره ب

، ، استخلاص ، التراص ، الخصائص ، اتاللكتين : الكلمات المفتاحية

اختبار التثبيط .

Glytaraldehyde

.Glytaraldehyde

Fetuin

.EDTA

.O, B, A

Moringa oleifera

Moringa oleifera

.Sephadex G-50

ABBREVIATIONS

CMoL: Coagulant M. oleifera Lectin

Con A: Concanavalin A

CRD: Carbohydrate Recognition Domain

FAO: Food and Agriculture Organization

Fuc: Fucose

HIV: Human Immunodeficiency Virus

Gal: Galactose

GalNAc: N-acetyl-D-galactosamine

GlcNAc: N-acetyl glucosamine

Man: Mannose

MO: Moringa oleifera

NeuNAc: N-acetylneuraminic

PNA: Peanut lectin

RBC: Red Blood Cell

Rh: Rhesus

SHA: Specific Hemagglutinating Activity

WGA: Wheat Germ Agglutinin

WSMoL: Water-Soluble M. oleifera Lectin

LIST OF FIGURES

Figure 01A: Hemagglutination assay for lectin detection…………………………………….6

Figure 01B: Inhubition of heamagglutination to assure lectin presence and specificity with

carbohydrate……………………………………………………………………………............6

Figure 02: Binding Selectivities of Plant Lectins. The plant lectins wheat germ agglutinin,

peanut lectin and phytohemagglutinin recognize different oligosaccharides……………….…7

Figure 03: Graphic representation of a monomer of Concanavaline A from Canavalia

ensiformis in complexe with the trimannosoide……………………………………………….8

Figure 04: Roles of lectins in microorganisms………………………………….………..…10

Figure 05: Agglutination tests of ABO blood group system……………………………..….15

Figure 06: ABO antigen specificity………………………………….………………….…...16

Figure 07: The distribution of Moringa oleifera in the World…….…………………….…..18

Figure 08: Diagram showing the extraction procedures of lectins from Moringa oleifera

seeds…………………………………………………………………………………………..23

Figure 09: Elution profile of the 0-50% ammonium sulfate precipitate on Sephadex G-50

column…………………………………………………………………………………….32

Figure 10: Bradford assay standard curve…………………………………………………....34

Figure11: Effect of temperature on the agglutinating activity………………….……………39

Figure12: Effect of pH on the agglutinating activity……………………………………………..43

LIST OF TABLES

Table 01: History of lectins …………………………………………………………….4

Table 02: Lectin specificity to monosaccharide………………………………………...7

Table 03: Animal lectins according to their structural characters………………….…...9

Table 04: Biological activities of lectins from different plant tissues…………………10

Table 05: Structural classification of plant lectins…………………………………….11

Table 06: Plant lectins with antimicrobial activity…………………………………….12

Table 07: Antigens and antibodies of the ABO system………………………………..15

Table 08: Plant lectins and their specificity in ABO system…………………………..16

Table 09: Classification of Moringa oleifera………………………………….………20

Table 10: Bioactive compounds in M. oleifera seeds………………………………….21

Table 11: Agglutination of the crude extract of Moringa oleifera seeds……………29

Table 12: Limit agglutination of extract M.oleifera seeds…………………………….30

Table 13: Protein content of different fraction of Moringa oleifera seeds…………….34

Table 14: Purification profile of Moringa oleifera seed lection……………………….35

Table 15: Inhibition test of Moringa oleifera seed lectin ……………………..………36

Table 16: Inhibition limit agglutination of fetuin with extracts M.oleifera seeds……37

Table 17: Effect of temperature on the agglutination test of Moringa oleifera………..38

Table 18: Effect of pH on the agglutination test of Moringa oleifera…………………40

LIST OF PHOTOS

Photo 01: A tree of Moringa oleifera………………………………………………….19

Photo 02: Aspects of Moringa Oleifera……………………………………………….19

Photo 03: Moringa oleifera seeds………………………………………..………….…22

Photo 04: The powder obtained from M.oleifera seeds………………………………..22

Photo05: The crude lectin extract at different temperature……………….………..….27

Photo06: Agglutination test of crude lectin source…………………………………….29

Photo 07: Limit of the agglutination test of the crude extract…………………………30

Photo 08: Limit of the agglutination test of extract M.oleifera seeds after

precipitation…………………………………………………………………………….31

Photo 09: The agglutination test of different fraction 20, 21, 22, 29 and 30…...……...33

Photo 10: Limit of the agglutination test of different fraction 20, 21, 22, 29 and 30….33

Photo 11: Inhibition test..................................................................................................35

Photo12: Inhibition limit test of Fetuin with M.O……………………………………..37

Photo13: Effect of temperature on the agglutination activity of Moringa oleifera……39

Photo14: Effect of pH on the agglutination test of Moringa oleifera………………….40

Photo15: Agglutination test with the ABO blood system……………………………...41

Photo16: Effect of EDTA on the agglutinating activity………………………………..43

Introduction

Introduction

1

Biomolecules essentially fulfill their function through continual recognition of

and binding to other molecules. Biomolecular recognition is therefore a phenomenon of

prominent importance. Proteins are a remarkable example in view of their capacity to

change shape. Several decades ago, the author of a well-known treatise on proteins

(Creighton, 1983) wrote that ... the biological function of proteins almost invariably

depends on their direct physical interaction with other molecules. (Pierre, 2014).

Amongst these proteins, our interest is ‘lectins’, a special class of proteins widely

distributed in nature, which selectively recognize and reversibly bind to carbohydrates

and glycoconjugates through their binding sites. (Santos et al., 2014). These proteins

were discovered towards the end of the 19th century and were referred to as

hemagglutinins due to their ability to agglutinate erythrocytes or phytoagglutinins

because they were originally found in extracts of plants. (Sharon and Lis., 2004).

Lectins have been powerful tools in preparative and analytical purposes in

biochemistry, cell biology, immunology, molecular biology, pharmacology and clinical

chemistry. (Maricel et al., 2004). They manifest a diversity of activities including anti-

insect activities, antitumor, immunomodulatory, antimicrobial and HIV-1 reverse

transcriptase inhibitor, which may find applications in many therapeutic areas. (Rabia

et al., 2013).

According to the Food and Agriculture Organization’s (FAO) report, about

70–80 % of the world’s population, especially in developing countries, relies on herbal

medicine to prevent and cure diseases (Ekor., 2014), and about 25 % of the synthesized

drugs are manufactured from medicinal plants (Pan et al., 2013).

Moringa oleifera Lam., a common marginal tropical tree in Africa, also known as

‘the miracle tree’ has become in a decade one of the new food and economic important

plant resources. (Atakpama et al., 2014). Various parts of the plant including roots,

leaves, and seeds possess various medicinal as well as nutritional values. (Stohs and

Hartman., 2015).

The broad application and variety of uses of lectins show the need to isolate

lectins from local and cheap sources since lectins are very expensive. One of the

Introduction

2

possible local sources of lectin is the seed of horseradish or malunggay (Moringa

pterygosperma syn. Moringa oleifera). (Maricel et al., 2004).

This present work was done to study the presence of lectins in this plant, which

has never been studied in Algeria in line with the extraction of lectins.

The main aim of the study is to evaluate the agglutinating activity of the lectins

extracted using rabbit erythrocytes, purification of the extract, study the effect of

thermal and pH treatment on the stability of this lectin, and detect its specificity using

the inhibition test by sugars.

Brief review on

Lectins

Brief review on Lectins

3

I. LECTINS

1. Definition

The word lectin has been derived from the Latin word “legere”, which means “to

select”, by William Boyd (Boyd and Shapleigh., 1954). This term was generalized to

embrace all sugar-specific agglutinins of non-immune origin, irrespective of source and

blood type specificity (Sharon and Lis., 1972). Lectins have the ability to bind

carbohydrates and the name “hemagglutinins” is used when the sugar specificity is

unknown.

Lectin is defined as a carbohydrate-binding protein of non-immune origin that

agglutinates cells or precipitates polysaccharides or glycoconjugates (Goldstein et al.,

1980). They can bind to the carbohydrate moieties on the surface of erythrocytes and

agglutinate the erythrocytes, without altering the properties of the carbohydrates.

(Rabia et al., 2013).

The ability to agglutinate cells distinguishes these proteins from other

macromolecules able to bind carbohydrates. In addition, their non-immune origin

differentiates them from anti-carbohydrate immunoglobulins that agglutinate cells.

(Santos et al., 2014).

More than a hundred of these molecules have been isolated from plants, viruses,

bacteria, invertebrates and vertebrates, including mammals. Lectins are a component of

traditional herbs such as dietary and medicinal plants. (Jasminka, 2015).

2. History of lectins

The occurrence in nature of erythrocyte-agglutinating proteins has been known

since the turn of the 19th century. (Sharon and Lis., 2004).

The study of lectins began with the work of Hermann Stillmark (1888) who, for

the first time, observed that seed extracts (Ricinus communis) could agglutinate red

blood cells. After this pioneer study, several theses and papers were published. (Renato

et al., 1991). Subsequently, H. Hellin, also at Tartu, demonstrated the presence of a

toxic hemagglutinin, abrin, in extracts of the jequirity bean (Abrus precatorius).

In 1919, James B. Sumner at Cornell University (Ithaca, New York), isolated

from jack bean (Canavalia ensiformis) a crystalline protein that he named concanavalin

A and in this way obtained a pure hemagglutinin for the first time.


4

However, nearly two decades passed before Sumner and Howell (1936) reported

that Concanavalin A agglutinates cells such as erythrocytes and yeasts and also

precipitates glycogen from solution. They further showed that hemagglutination by

Concanavalin A was inhibited by sucrose, demonstrating for the first time the sugar

specificity of lectins. (Sharon and Lis., 2004). Table 01 shows some milestones of

lectinology.

Table 01: History of lectins. (Renato et al., 1991).

Year Scientists Discovery

1884 Warden and Waddel/

Bruyllants and venneman.

Toxicity in Abrus precatorius seed extracts.

1886 Dixson. Toxicity in Ricinus communis seeds

extracts.

1888 Hermann Stillmark. Hemagglutinating activity in Ricinus

communis.

1890 P Erlich. Use of abrin and ricin in immunological

research.

1908 K Lansteiner and H

Raubitsheck.

Different hemagglutinating properties in

various seeds extracts.

1919 J B Sumner. Crystallization of concanavalin A.

1936 J B Sumner.

S F Howell.

Lectins demonstrated to bind sugar

Concanavalin A precipitates glycogen for

some human blood group antigens.

1940 W C Boyd, R M Reguera and

K O Renkonen.

Specificity of some lectins for some human

blood group antigens.

1954 W C Boyd and E Shyleigh. The name lectin proposed instead of

hemagglutinin.

1960 P C Nowell. Lectin from Phseolus vulgaris found to be

mitogenic to resting lymphocytes.

1960 J C Aub. Lectins preferentiallt agglutinate malignant

cells.


5

1974 G Ashwell and

A G Morell.

First mammalian lectin identified:

hepatocyte asialoglycoprotein receptor

specific for terminal galactose in serum

glycoprotiens.

1976 Y Reisner. Peanut agglutinin discriminates cortical from

medullary cells in mice.

1977 Ofek et al. Role of bacteria lectins in infection.

1980 Pusztai. Interaction of Phaseolus vulgaris lectins

with intestinal wall.

1981 Reisner et al. Use of lectins in bone marrowt

transplantation.

1984 Yajko et al. Combined use of lectin and enzyme in

clinical identification of micro-organisms.

1987 Harban-Mendoza et al. Control of root-knot nematodes by lectins.

1988 De Oliveira et al. Lectin and pancreas hyperthrophy.

1989 Diaz et al. Root lectin as a specificity determinant in

the Rhizobium-legume symbiosis.

1990 Yamauchi and Minamikawa. Con A expression in Escherichia coli cells.

3. Detection and specificity

The simplest way to detect a lectin is to examine its ability to agglutinate

erythrocytes or to precipitate glycoconjugates using the hemagglutination test. (Figure

01A). (Goldstein et al., 1980).

Lectins are, in most cases, di- or multivalent and able to interact with

carbohydrates or glycoproteins in solution or linked to cell membranes and their binding

sites interact with cells forming various reversible linkages. Because of this ability,

lectins are easily detected through agglutination assays. (Santos et al., 2014).

For a better characterization of the lectin however, it is essential to determine

whether it is specifically inhibited by mono- or oligosaccharides. This specificity is


6

usually determined by the hapten inhibition techniques, comparing the sugars on the

basis of the minimum concentration to inhibit hemagglutination or precipitation

reactions as shown in Figure 01B. (Renato et al., 1991).

Figure 01A: Hemagglutination assay for lectin detection. (Santos et al., 2014).

Figure 01B: Inhibition of hemagglutination to assure lectin presence and specificity

with carbohydrate. (Santos et al., 2014).

Depending on carbohydrate specificity, major lectins are divided into mannose

binding lectins, galactose/N-acetylgalactosamine binding lectins, N-acetylglucosamine

binding lectins, N-acetylneuraminic acid binding lectins and fucose binding lectins as

shown in Figure 02. (Jasminka, 2015).


7

Figure 02: Binding Selectivities of Plant Lectins. The plant lectins wheat germ

agglutinin (WGA), peanut lectin (PNA) and phytohemagglutinin recognize different

oligosaccharides. (Jasminka, 2015).

Table 02: Lectin specificity to monosaccharides. (Sharon, 2007).

Sugar Lectins

Mannose (Man) Allium sativium ; Canavaliaensiformis ; Crocus

sativus ; Diocleandiflora E.coli type 1 fimbriae ;

ERGIC-53 . Galanthusnivalis ; MBLs of animals ;

Pisumsativum.

Fucose (Fuc) Aleuriaaurantia ; Anguilla Anguilla ; Lotus

tetragonolobus ; Pseudomonas aeruginosalectin 2 ;

Ulexeuropaeuslectin 1 ; Ulvalactuca ;

Chromobateriumviolaceumlectin.

Galactose (Gal) /

N-acetylgalactosamine

(GalNAc)

Arachishypogaea ; Coprinuscinereus ;

Entamoebahistolytica ; Erythina corallodendron ;

Dolichosbiflorus ; Glycine max ;

Griffoniasimplicfoglia lectin 1 ; helixpomatia ;

HygrophorushypotheJus ; Phaseolus limensis

N-acetylglucosamine

(GlcNAc)

Conglutinin ; Griffoniasimplicifoglialectin 2 ;

Tachylectin-2 ; Triticum aestivum ;

Ulexeuropaeuslectin 2; Psathyrellavelutina.

N-acetylneuraminic

)NeuNAc(

Achatinafulica ; Cancer antennarius ;

Hericiumarinaceum, Homarusamericanuslectin 1 ;

Limaxflavus


8

4. Structural characteristics

Specificity of lectins to carbohydrates, determined by three-dimensional structure

of their binding sites, shows a conserved amino acid profile within families of lectins.

(Peumans and Van Damme., 1998).

Lectins can have associated metal ions and interactions coordinated by water

molecules and carbohydrates (Sharon and Lis., 2002). These proteins can present 2 to

12 sites of interaction, depending on the nature of the molecule and oligomerization

state. (Balzarini, 2006).

Structural differences occur in lectins from the primary structure to the last degree

of molecular organization; they may differ in amino acid sequence, change in the

number of subunits, and in the nature of the polypeptides. Interactions between the

subunits seem to play a dominant role in the stability of these proteins. (Mitra et al.,

2002).

Figure 03: Graphic representation of a monomer of Concanavaline A from

Canavalia ensiformis in complexe with the trimannosoide. (Lenka, 2006).

5. Production of lectins

Lectins are found in nature. A large number of lectins or hemagglutinins have

been purified from different organisms.

a. Animal lectins

Lectins found in animals are most often found to aid in cell interactions. (Berg et

al., 2002). They seem to be involved in the mechanisms of endocytosis, intracellular

translocation of glycoprotein, binding to glycoconjugates, apoptosis processes and


9

defense against microorganisms, regulating the processes of cell adhesion and migration

as well as in binding of bacteria to epithelial cells.

In vertebrates, there are two classes of lectins, based on their location: integral

lectins of membranes and soluble lectins present in intra and intercellular fluids

(Santos et al., 2014).

Gabius divided animal lectins into five main groups according to their structural

characters. (Gabius, 1997).

Table 03: Animal lectins according to their structural characters. (Gabius, 1997).

Lectin Characteristic

C-type lectin Depend on the presence of Calcium ions to bind

carbohydrates.

Conserved carbohydrate recognition domain (CRD).

I-type lectins CRD similar to immunoglobulins.

Galectins/ S-type lectins Thiol dependent.

CRD similar to B-galactosides.

Pentranxins Many subunits producing pentameric lectins.

b. Lectins from fungi, bacteria and viruses

Lectins in microorganisms appear to play several important roles, like the

interaction of host cells, recognition in immunological processes, phagocytosis, and cell

adhesion as shown in Figure 04. (Ponchel and Irache., 1998).

Fungal lectins, as in animals, are classified according to their structure, such as the

lectin from Coprinopsis cinerea, related to galectins, a lectin class characterized by

many conserved residues (Wälti et al., 2008).

Likewise, fungal lectins, as with plant lectins, can be classified according to their

binding specificity: to mannose (Francis et al., 2011), arabinose (Wang and Ng.,

2005), acetyl-Dgalactosamine (Chumkhunthod et al., 2006), melibiose, xylose (Zheng

et al., 2007), or lactose (Liu et al., 2008).


10

Figure 04: Roles of lectins in microorganisms. (Imberty and Varrot, 2008).

c. Plant lectins

Plants are a rich source of lectins and serve as the main source for isolation and

analysis of these molecules. Lectins in plants play important roles such as acting as a

reserve of nitrogen, as protein specific recognition factors and may also be involved in

defense mechanisms against pathogenic microorganisms, insects and predators due to

their toxicity. (Ripoll et al., 2003).

Lectins are present in various plant tissues and have different biological

properties.

Table 04: Biological activities of lectins from different plant tissues. (Santos et al.,

2014).

Tissues Biological activities

Seeds Anticoagulant and antiplatelet aggregating properties;

coagulant, mitogenic, antibacterial, antifungal and antitumor

activities.

Bark Antifungal and insecticidal activities.

Heartwood Termiticidal activity.

Stem Antiviral and apoptosis-inducing activities.

Leaves Antiviral, antibacterial and antifungal activities.

Fruits Mitogenic, antiviral activities.


11

Roots Antifungal and termiticidal activities.

Tubers Insecticidal and antitumor activities.

Bulbs Photolytic activity.

Rhizomes Antiproliferative, immunostimulatory, antiviral, antifungal

and antitumor and apoptosis-inducing activities.

There are four main, distinct classes of plant lectins based on their overall structure.

Merolectins, with only one CRD, are small monovalent proteins with a single

polypeptide and are incapable of precipitating glycoconjugates or agglutinating

cells.

Hololectins are lectins exclusively with CRDs, in which two or more of them

are identical or very similar. This class includes all lectins that have multiple

binding sites and are able to agglutinate cells or precipitate glycoconjugates.

Chimerolectins, the result of protein fusion, contain one CRD and another

unrelated domain acting independently. (Kelany et al., 2012).

Superlectins consist of molecules with two or more distinct CRDs. (Van

Damme et al., 1998).

Table 05: Structural classification of plant lectins. (Van Damme et al., 1998).

Merolectins Hololectins Chimerolectins Superlectins

Plant lectins can also be classified according to the specificity of their interactions

with mannose/glucose (Bari et al., 2013), mannose/maltose, galactose/N-

acetylgalactosamine, N-acetylglucosamine/ N-acetylglucosamine, galactose (Wong et

al, 2006), mannose, fucose and sialic acid among others.


12

6. Biological properties and applications

Lectins stand out in biotechnology due to their many applications and other

potential uses that are being evaluated daily and studied by researchers in lectinology.

Lectins and their characteristic properties, mainly due to their ability to bind

glycoconjugates, stand out as important tools in research covering various areas of

science, especially in biochemistry, cellular and molecular biology, immunology,

pharmacology, medicine and clinical analysis.

Lectins have a variety of effects on cells, such as agglutination, mitogenic

stimulation, redistribution of cell surface components, modifying the activity of

membrane enzymes, inhibition of bacterial and fungal growth, cell aggregation,

toxicity, immunomodulation, among others. (Santos et al., 2014).

6.1. Lectins against microorganisms

Many human pathogens utilize cell surface glycans as either receptors or ligands

to initiate adhesion and infection (Oppenheimer et al., 2008). Escherichia coli (E.coli),

for example, binds to host mannosides, while influenza virus binds to host sialic acids

(Mukhopadhyay et al., 2009). Cytotoxic effects of lectins may be revealed by

antitumoral and antiviral activities and also by deleterious effect on microorganisms

(Table 06). (Rabia et al., 2013).

Lectins, especially those with specificity to mannose or N-acetyl-glucosamine,

have a remarkable anti-HIV (Human Immunodeficiency Virus) activity as shown in cell

culture assays (Molchanova et al., 2007).

Table 06: Plant lectins with antimicrobial activity. (Rabia et al., 2013).

Plant (tissue) Lectin specificity Antimicrobial activity

Araucaria angustifolia

(seed)

GlcNAc Clavibacter michiganensis,

Xanthomonas axonopodis pv.

Passiflorae.

Talisia esculenta (seeds) Man Colletotrichum lindemuthianum,

F. oxysporum, S. cerevisiae

Phaseolus coccineus

(seeds)

Sialic acid Helminthosporium maydis,

Gibberalla sanbinetti, R.

solani,Sclerotinia sclerotiorum


13

6.2. Lectins and insects

Lectins, beyond their effects on cells, also have the ability to act as insecticidal

molecules against a variety of species. The mechanisms of insecticidal action of lectins

are unknown, although entomotoxic activity seems to depend on the carbohydrate

recognition property that they exhibit (Paiva et al, 2012). In general, lectins able to bind

N-acetyl-D-glucosamine with affinity to chitin have insecticidal properties.

Arisaema helleborifolium lectin exhibited anti-insect activity towards the second

instar larvae of B. cucurbitae (Kaur et al., 2006).

6.3. Lectins and cytochemistry/histochemistry

The glycan moieties covering cell surfaces are involved in many physiological

and pathological processes related to cell. Disturbances in cell environment related to

diseases frequently triggers changes in glycans such as fucosylation, abnormalities in

glycan structures and uncommon glycans. (Svarousky and Joshi., 2014).

The ability of lectins to bind carbohydrates is useful in investigating the changes

in the expression of glycans on cells in tissue markers. (Roth., 2011).

Generally, lectin histochemistry uses peroxidase-conjugated lectin followed by

addition of diaminobenzidine and hydrogen peroxidase for visualization of binding.

An example is Parkia pendula lectin conjugated to horseradish peroxidase was

evaluated as a histochemical marker for characterization of meningothelial tumor tissue.

(Beltrao et al., 2003).

6.4. Lectins and anti-tumor activity

Tumour cell surfaces vary in composition of glycoconjugates in comparison to normal

cells. (Eckhardt and Goldstein.,1983). Evidence is now emerging that lectins are

dynamic contributors to tumor cell recognition (surface markers), cell adhesion and

localization, signal transduction across membranes, mitogenic stimulation,

augmentation of host immune defense, cytotoxicity, and apoptosis. (Hamid et al.,

2013).

(Liu et al., 2009) reported that ConA , a typical legume lectin with a mannose/glucose-

binding specificity, was reported to induce apoptosis in murine macrophage PU5-1.8

cells through clustering of mitochondria and release of cytochrome c.

Brief review on

ABO blood system

Brief Review on ABO system

14

II. ABO SYSTEM

1. History

It was not until the year 1900, when Karl Landsteiner at the University of

Vienna, discovered why some blood transfusions were successful while others could be

deadly.

Landsteiner discovered the ABO blood group system by mixing the red cells and

serum of each of his staff. He demonstrated that the serum of some people agglutinated

the red cells of other. From these early experiments, he identified three types, called A,

B and C (C was later to be re-named O for the German “Ohne”, meaning “without”, or

“Zero”, “null” in English). The fourth less frequent blood group AB was discovered a

year later. (Dariush et al., 2013).

After this discovery, gradually from 1927, other blood groups were also

discovered and reported including the Rhesus (Rh) blood group that was discovered by

Landsteiner together with his American colleague Alexander Wiener in 1940, 1941.

2. The ABO system

ABO remains the most important in transfusion and transplantation among all the

blood group systems. (Sano et al., 2012). The blood type is defined by oligosaccharide

structures, which are specific to the antigens, thus, blood group antigens are secondary

gene products, while the primary gene products are various glycosyltransferase enzymes

that attach the sugar molecules to the oligosaccharide chain. (Ewald and Susan., 2016).

The gene that determines human ABO blood type is located on chromosome 9 (9q34.1)

and is called ABO glycosyltransferase. (Ranadhir et al., 2014).

The system comprises complex carbohydrate structures that are biosynthesized by the A

and B transferases encoded by the A and B genes, respectively. (Schenkel, 2000). He

also recognized that the sera of individuals lacking one or both antigens usually contain

anti-A and/or anti-B is agglutinins. In the course of further investigations many

subgroups and phenotypes have been detected which are defined both by quantitative

differences in antigen content as well as by a characteristic tissue distribution of the

antigens. (Brunner, 2000).


15

Table 07: Antigens and antibodies of the ABO system. (Bailly et al., 2015).

Agglutination of red cells arises because one individual’s serum contains

naturally occurring antibodies against ABO antigens that are missing from that

individual’s own red cells, but which are present on the cells of another: e.g. group A

individuals have Anti-B that agglutinates group B cells. (Figure 05) (Anatole and

Marcela., 1996).

Figure 05: Agglutination tests of ABO blood group system. (Stelling, 2008).

3. The Rhesus system

Rhesus system is the second most important blood group system after ABO.

(Westhoff., 2004). Red blood cell surface (RBCs) of an individual may or may not

have an Rh factor or immunogenic D‑antigen. Accordingly, the status is indicated as

either Rh-positive (D‑antigen present) or Rh‑negative (D‑antigen absent). In contrast to

the ABO system, anti‑Rh antibodies are, normally, not present in the blood of


16

individuals with D‑negative RBCs, unless the circulatory system of these individuals

has been exposed to D‑positive RBCs. (Ranadhir et al., 2015).

4. Antigen Structures of blood groups of the ABO system

ABO blood types are determined by a cell surface marker that identifies this as

belonging to self or to that individual. These cell surface markers are characterized by a

protein or lipid that has an extension of a particular arrangement of sugar. Figure 06

shows the arrangement of sugars that determine each of the A, B, and O blood types.

(Goldsby et al., 2000).

It should be noted that each is identical except that types A and B have an

additional sugar; N-acetylgalactosamine for A, and Galactose for B. (Daniel, 2008).

Figure 06: ABO antigen specificity. (Daniel, 2008).

5. Plant lectins and their specificity in ABO system

The presence of carbohydrates on the outer surface of the cell membranes of

erythrocytes (Figure 06) facilitates the blood group detection by lectins specific to the

carbohydrates.

Table 08: Plant lectins and their specificity in ABO system. (Paolo and Richard.,

1992).

Lectin from

(common name)

Blood type Monosaccharide recognized


17

Griffonia simplicifolia

(GS-I)

A,B α -D-Gal, α -D-GalNAc


(GS-IB4)

B α -D-Gal


(GS-IA4)

A α -D-GalNAc

Helix pomatia (HPA) A α -D-GalNAc

Ptilota plumose B α -D-Gal

Anguilla anguilla O α –L-Fuc

Ulex europaeus (UEA-I) O α –L-Fuc

Laburnum alpinum (LA-I) O (GlcNAc) 2-3

Laburnum alpinum (LA-II) O Gal

Brief review on

Moringa oleifera

Brief Review On Moringa oleifera

18

III. MORINGA OLEIFERA

1. Generalities

Moringa oleifera Lam. (Moringaceae) is a medium-sized tree autochthonous from

northeastern India and widely distributed worldwide throughout the tropics and

subtropics (Teixeira et al., 2012). The distribution of M. oleifera in the world is

outlined in Figure 07.

It is recognized as a vibrant and affordable source of phytochemicals, having

potential applications in medicines, functional food preparations, water purification, and

biodiesel production. (Ramesh et al., 2016).

Due to its rich source of certain macro and micro nutrients which are of great

importance, Moringa oleifera is referred to as a ‘Miracle tree’. (Adewumi and

Samson., 2016).

It is in fact said to provide 7 times more vitamin C than oranges, 10 times more

vitamin A than carrots, 17 times more calcium than milk, 9 times more protein than

yoghurt, 15 times more potassium than bananas and 25 times more iron than spinach

(Lakshmipriya, 2016).

Figure 07: The distribution of Moringa oleifera in the World.

(www.outline-world-map.com).

2. Description of Moringa oleifera plant

Moringa oleifera Lam is a tropical deciduous perennial fast growing plant

dicotyledonous tree which can reach 7-12 m of height, sometimes even 15 m (Larissa

et al., 2013).

http://www.outline-world-map.com/


19

The stem is brittle with a corky, whitish-gray bark, with drooping branches, pale

green and bipinnate or more commonly tripinnate leaves (30–60 cm long) with

opposite, ovate leaflets (Pandey et al., 2011).

The Moringa tree grows best in the temperature range of 25–35°C, under direct

sunlight, at an altitude of 500 m, and in slightly acidic to alkaline soil (pH 5.0–9.0);

although it can tolerate excess temperature, up to 48°C, frost in winter, altitude, and a

wide variety of soil conditions. (Ramesh et al, 2016).

Photo 01: A tree of Moringa oleifera. (Alessandro et al., 2015).

Photo 02: Aspects of Moringa Oleifera, A: tree, B: seedpods, C: flowers, D: seeds.

(Lakshmipriya, 2016).


20

3. Taxonomic classification

Table 09: Classification of Moringa oleifera. (Garima et al., 2011).

Kingdom Plantae

Sub kingdom Tracheobionta

Super Division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Dilleniidae

Order Capparales

Family Moringaceae

Genus Moringa

Species Oleifera

4. Properties of Moringa oleifera

Every part of the plant (leaves, seeds, roots, flowers, bark, fruits and pods) is a

rich storehouse of a number of nutrients such as proteins, fibre and minerals that play an

important role in nutrition. (Lakshmipriya et al., 2016).

Several extractions also tested positive for the presence of phytochemicals like

unique glucosinates, flavonoids, and phenolic acids, carotenoids, tocopherols and

polyunsaturated fatty acids.

These phytochemicals of M. oleifera have shown antidyslipidemic, anthelmintic,

antihyperglycemic, anti-inflammatory, antimicrobial, antioxidant, antiproliferative, anti-

ulcer, antiurolithiatic, and hepatoprotective properties. (Garima et al., 2011).


21

Table 10: Bioactive compounds in M. oleifera seeds.

Compounds References

Alkaloids

Moringine

)Kirtikar et al., 1975(.

Flavonoids

Catechin

Epicatechin

Quercetin

Kaempferol

)Govardhan et al., 2013(.



)Singh et al., 2009(.

Phenolic acids

Gallic acid

P-Coumaric acid

Ferulic acid

Caffeic acid

Protocatechuic acid

Cinnamic acid

Ellagic acid

)Singh et al., 2009, Govardhan et al., 2013(.






)Singh et al., 2009(.

Glycosides

4-(2/3/40-O-acetyl-α-L-

rhamnopyranosyloxy)benzyl

glucosinolate

)Maldini et al., 2014(.

Shelled M. oleifera seeds have the property to decontaminate arsenic from water

and can be used as domestic and environment-friendly safe technology (Kumari et al.,

2006) due to their coagulant properties.

Two lectins deemed coagulant M. oleifera lectin (cMoL) and water-soluble M. oleifera

lectin (WSMoL) are among the coagulant proteins present in the seeds (Santos et al.,

2009; Ferreira et al., 2011).

M. oleifera seeds contain 33–41 % (w/w) oil, known as ‘‘ben oil’’, because of the

contents of behenic acid (C22, docosanoic acid, &7 % w/w), which possesses

significant resistance to oxidative degradation (Rashid et al., 2008).

Material and

Methods

Material And Methods

22

MATERIALS AND METHODS

1. Plant material

Mature M. oleifera (Family: Moringaceae) seeds were brought from the gardener

of Algeria. We used the seeds to extract lectins.

Photo 03: Moringa oleifera seeds.

2. Methods

A. Preparation of M. oleifera

Drying: The seeds of Moringa oleifera were dried at room temperature for

7 days.

Grinding: The dried seeds were then ground using a mortar and pestle till a

powder was obtained.

Photo 04: The powder obtained from M.oleifera seeds.


23

B. Extraction of lectin from M. oleifera

Moringa oleifera seed powder was treated with phosphate buffered saline (PBS,

10mM, pH 7.4) at a ratio of 1:10(w/v). The extraction of proteins was continued by

stirring the mixture for 24 hours under refrigeration.

The extract was filtered, using filter paper, then centrifuged at 6300xg for 30 minutes.

The residue was disposed of while the supernatant was recovered and used as the crude

lectin source.

Figure 08: Diagram showing the extraction procedures of lectins from Moringa oleifera

seeds.

The powder of plant M.oleifera.

M.oleifera was put in buffer for

24 hours under refrigeration.

Filtration of liquid of M.oleifera.

Centrifugation at 6300xg for 30

min.

Supernatant

Crude extract


24

C. Preparation and treatment of rabbit erythrocytes

This was carried out according to Kumar and Rao., 1986. Rabbit blood was

obtained from the laboratory-animal facility at the University of the Mentouri Brothers,

Constantine 1. It was collected in a 4 ml heparin tube. The mixture was centrifuged at

3000xg for 10 min at 4°C, and the residue obtained is washed 3 times with NaCl

solution 0.9%, followed by a centrifugation after each washing.

This precipitate was treated by dissolving it in a solution of 1% glutaraldehyde,

centrifuged and washed two times.

D. Testing for Agglutinating activity

Hemagglutinating activity (HA) was assessed in microtiter plates according to

(Correia and Coelho., 1995). 50μl of the extract is added to 50μl of glutaraldehyde

treated rabbit blood. The plate was allowed to stand for 30 minutes and was observed

for agglutinating activity with naked eye as a gelatinous form.

A control test was performed by adding 50µl of erythrocytes to 50µl of PBS.

E. Limit of the agglutinating activity

This test was performed to determine the highest dilution that causes

agglutination, hence the agglutinating strength of the lectin. . HA, the reciprocal of the

highest dilution of the sample promoting full agglutination of erythrocytes, was defined

as one hemagglutination unit (Chumkhunthod et al., 2006).

It was performed according to the method of serial double dilution described by

Eretan O., 2004.

50μl of PBS was put in each well of one horizontal line of the microtitre plate,

then 50μl of the crude extract was added to the first well, and was serially diluted two-

fold along the line. 50μl of fixed erythrocytes was added to each well. The agglutination

activity was observed after incubation of the plate for 1 hour at room temperature.


25

1. Further Purification

a. Salting-out by Ammonium sulfate

Protein solubility is affected by ions. At a very high ionic strength, protein

solubility decreases as ionic strength increases in the process known as ‘salting-out’.

(Duong and Gabelli., 2014).

The crude extract was precipitated using ammonium sulfate. The amount of

(NH4)₂SO4 to be added was based on the monogram table by Cooper (1977).

Ammonium sulfate was added to the crude PBS extract in two fractions 0-50%, and 50-

80% over a 30min period with constant stirring at 4°C. The precipitate was separated

by centrifugation at 8000xg for 20 min at 4°C and was kept at 0°C while the

supernatant was further saturated with (NH4)₂SO4 to completely precipitate any

remaining protein in solution.

Likewise, the precipitate was collected by refrigerated centrifugation. It was dissolved

in minimum amount of PBS (pH 7.4).

The precipitates are then tested for agglutinating activity to obtain the fraction with

the maximum protein precipitate.

The same procedure as above is followed to test the limit of the agglutinating

activity, only changing the crude PBS extract with the precipitate.

b. Dialysis

This step is done to permit desalting of the precipitate. The precipitated protein (0-

50%), the most active fraction, is submitted to dialysis (membrane of exclusion limit

12kDa) against 1litre of PBS (0.01M, pH 7.4). The reaction takes place over night

under agitation at 4°C.

c. Gel filtration chromatography

The fraction after dialysis was subjected to molecular sieving through the

equilibrated Sephadex G-50 column eluted with PBS (pH 7.4). 5mL of the sample was

loaded to the column and a 2 mL fraction of the eluted sample was collected at a flow

rate of 0.1ml/sec by gravity. The eluted fractions were monitored for

spectrophotometric absorbance at 280nm.


26

The fractions that showed a peak in the optic density were subjected to an

agglutination test.

2. Protein determination

The protein contents of content of the lectin samples obtained during the

purification process was determined according to the method of Bradford et al (1951)

using bovine serum albumin as standard.

A volume range of the standard (0, 10, 20, 30, 40, 50, 60, 70, 80, 100 µl) is

prepared in glass test tubes from a solution of BSA 1mg/ml, each with three repetitions.

These volumes are then adjusted to 100µl with distilled water.

100µl of PBS, the extract and of each of the fractions are also prepared. 4ml of the

Bradford solution is added to each of the test tubes, and left for 10 minutes for reaction.

The absorbance of each is measured at 595nM against the empty solution.

3. Characterization of the lectin

a) Inhibition test

This test permits us to know the potential ligands (sugars and glycoproteins) of

the lectin. The active fraction (0-50%) is diluted with PBS, pH 7, 4. (To 100µl of the

fraction is added 3100µl of the buffer.)

Different carbohydrates and glycoproteins were prepared with a concentration of

400mM with PBS.

25µl of PBS was deposited in each well. 25µl of the ligand was added to the first

well, and then a series of double dilutions in the next 2 wells. 25µl of the diluted active

fraction was added to each well and incubated for 30 minutes at room temperature.

After incubation, 50µl of rabbit erythrocytes are added to the wells and incubated for

1hour at room temperature and the agglutinating activity was observed with a naked

eye.


27

b) Effect of temperature on the stability of the protein

The crude lectin extract was subjected to various temperatures, 20°C, 30°C, 40°C,

50°C, 60°C and 70°C, 80°C, 90°C, 100°C for 30 min.

2ml of the extract was incubated at various temperatures, 30°c, 40°c, 50°c, 60°c, 70°c,

80°c, 90°c and 100°c in a water bath for 30 min, and then cooled at room temperature.

The heat-treated lectin was tested for agglutination using treated rabbit blood.

Photo 05: The crude lectin extract at different temperature.

c) Effect of pH on the agglutinating activity

The effect of pH on the agglutinating activity of the dialyzed fraction (30-50%) is

carried out on a range pH 2-10. (2, 3, 4, 5, 6, 8, 10).

2ml of the fraction is placed in a dialysis membrane and submerged in a beaker

containing the buffer solution of the specific pH medium. The reaction takes place

under agitation, at 4°C for 24 hours. It is then neutralized in 300ml of PBS, (10mM, pH

7.4) for 4 hours at 4°C.

The limit of the agglutination is then tested as in the previous tests.

d) The agglutination test with ABO system

The test was carried out to deduce the specificity of the blood group extract; it

was carried out on the human red blood cell antigens belonging to the ABO blood

group system using the erythrocytes of the different blood group (A, B and O).


28

e) Effect of EDTA on the agglutinating activity

50µl of EDTA at different concentrations (20mM, 30mM, 40mM, and 50mM)

was added to 50µl of the 0-50% fraction and incubated for one hour to allow reaction to

take place.

The four mixtures were tested for agglutinating activity, (50µl of PBS in all wells,

50µl of the mixture in the first well, then a series of double dilution in the rest of the

wells, and then 50µl of glutaraldehyde treated rabbit erythrocytes.

Two control tests were performed, a positive control test, where the agglutinating

activity is tested using the fraction without EDTA and a negative control test in which

we do not add any lectin containing fraction.

Results and

Discussion

Results and discussion

29

RESULTS AND DISCUSSION

1. Agglutination test of the crude extract

Agglutination is defined as the formation of clumps of cells or particles and

lectins have the ability to agglutinate erythrocytes.

Most lectins are able to interact with red blood cells via to the interaction between

membrane carbohydrates. If the erythrocyte was placed in a well, the natural

sedimentation leads to a deposit of the red cells at the bottom of the well of the

microtiter plate. The addition of the crude extract allows the formation of a network

between erythrocytes and lectins. These interactions form a homogeneous gelatinous

suspension; this corresponds to the phenomenon of agglutination.

Table 11: Agglutination of the crude extract of Moringa oleifera seeds.

Plant Agglutination test

Moringa oleifera (seeds) +++

+++: Presence of agglutination.

Test Front view Back view

Agglutination

with MO

extract

Control test

Photo 06: Agglutination test of crude lectin source.

A positive result is observed as a layer covering the bottom of the well of the

microtiter plate, while a negative result is indicated by precipitation of the erythrocytes

at the bottom of the wells and a red point is observed.

This confirms the presence of lectins in the saline crude extract and their ability

to agglutinate glutaraldehyde-treated rabbit erythrocytes.


30

Lubag (1988) and Pahm (1990) previously reported the presence of

agglutination factors in the seeds.

2. Limit of agglutination test

Obtaining the positive result was a go-ahead to test for the limit of this positive

agglutination test. Agglutination activity is expressed as the highest dilution ratio for

which agglutination was observed.

Table 12: limit agglutination of extract M.oleifera seeds.

1/2

1/4

1/8

1/16

1/32

1/64

1/128

1/256

1/51

2

1/10

24

1/20

48

1/40

96

M.oleifera +++ +++ +++ +++ +++ ++ ++ + - - - -

+++: Very strong agglutination, ++: Strong agglutination, +: Low agglutination, - : absence of

agglutination.

Titer

value

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

Front

view

Back

view

Control test

Photo 07: Limit of the agglutination test of the crude extract.

These results indicated the diminution of the agglutination activity with

increased dilution along the microtiter plate and agglutination stops at the titer value of

1/128.

Titer

value

Extract


31

3. Purification of the crude lectin extract

a) Salting-out

Lectins were separated from other substances co-extracted by PBS in the crude

extract by precipitating it with ammonium sulfate. Ammonium sulfate was chosen as

precipitating agent in this study due to its high solubility in water and produces high

ionic strength. Increase in ionic strength decreases the protein solubility. (Scopes,

1994).

The crude extract was subjected to different levels of (NH₄)₂SO₄ to be partially

purified. Agglutination was observed by the two dissolved precipitates collected at 0-

50% and 50-80% saturation.

The photo below show that the 0-50% (NH₄)₂SO₄ precipitate exhibited much

more agglutination even at the highest dilution more than the 50-80% (NH₄)₂SO₄

precipitate.

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

0-50%

50-80%

Control test

Photo 08: Limit of the agglutination test of extract M.oleifera seeds after

precipitation.

The two fractions indicated no agglutination in the ⅟₂ titer. This is due to the

high concentration of the lectins which instead of agglutination causes the rupture of the

erythrocytes by osmosis.

Titer

value

Fraction


32

In the 0-50% fraction, agglutination is observed up to the ⅟₅₁₂ dilution while in

the 50-80% fraction, the agglutination stops at the ⅟₂₅₆ dilution titer.

Conclusion: The 0-50% fraction contains the highest concentration of the protein

hence the most active to use for the rest of the tests.

b) Gel Chromatography

Gel filtration using Sephadex G-50 can resolve proteins with molecular weights

between 1.5 to 30KDa. (Ciaran et al., 2011).

The figure below shows the elution profile of the dialysed 0-50% ammonium

sulfate fraction. Two peaks were visible after reading the absorbance of the 55 fractions

collected (2ml per tube) and the fractions 20, 21, 22, 29 and 30 which fall on the first

and second peak showed agglutinating activity.

The seeds of Moringa oleifera may be containing two different lectins.

Figure 09: Elution profile of the 0-50% ammonium sulfate precipitate on Sephadex

G-50 column.

Agglutination activity of purified fractions

When tested for the agglutinating activity we find;

The first peak (F20, 21 and 22) exhibited the maximum lectin activity.

Tube


33

While the second peak (F29, 30, 31 and 32) showed a decreased lectin activity.

Control test F20 F21 F22

F29 F30 F31 F32

Photo 09: The agglutination test of different fractions.

The fractions with the highest agglutinating activity (20, 21, 22, 29, and 30) were

subjected to an agglutination test limit and the results are shown below;

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

20

21

22

29

30

Photo 10: Limit of the agglutination test of different fraction 20, 21, 22, 29, 30.

F20 showed the highest agglutinating activity up to a dilution of ⅟₆₄, followed by

F21 and F22 both having an agglutination activity up to the ⅟₃₂ dilution well, and then

F29 and F30 which only showed an agglutinating activity up to ⅟₄.

Titer

value

Fraction


34

4. Protein determination

Basing on the Bradford protein assay standard curve produced using BSA and its

linear regression equation; we were able to determine the quantity of proteins in each

fraction (the crude extract, the 0-50% fraction and the fraction after chromatography).

Figure 10: Bradford assay standard curve.

Table 13: Protein content of different fractions of Moringa oleifera seeds.

Fraction Absorbance Protein content (µg/ml) x10

Crude extract 0.787 77.87

0-50% fraction 0.827 83.2

Fraction after

chromatography

0.529 43.47

BSA .10¯¹ µg/ ml


35

Table 14: Purification profile of Moringa oleifera seed lection.

Fraction Total

volume

(ml)

Total

protein

(mg)

Total

activity

(HU)

SHA

(HU/mg)

Purification

fold

Yield

%

Crude

extract¹

500 38.935 128 3.288 1 100

0-50%

(NH₄)₂SO₄

fraction

50 4.16 512 123.08 37.433 400

G-50 Gel

filtration

4 0.1739 64 368.028 111.931 50

¹, 50g of seed powder were used, HU is the minimal concentration of protein required to cause visible

agglutination of a 4% glutaldehyde treated rabbit erythrocytes.

The protein was purified by 111.931 fold and with 50% yield.

5. Characterisation of the lectin

a. The inhibition test

The inhibition test was performed with certain saccharides to determine the

specificity of the carbohydrate extracts. Inhibition would occur if the sugar-lectin

interaction is stronger than the erythrocyte-lectin attraction and would consequently

form a distinct point of the erythrocyte at the bottom of the well.

Fetuin Other Sugars Control test

Photo 11: Inhibition test.


36

The agglutinating activity of the lectin was only affected by fetuin, a type O

glycoprotein while the other sugars showed no inhibition effect and showed the same

result as the control test in which no ligand was added.

The non-inhibition of agglutination by the simple sugar molecules may be due to

the fact that the lectin binding sites are non-specific for the sugar anomers. (Lacsamana

and Merca., 1994).

Table 15: Inhibition test of Moringa oleifera seed lectin.

Sugar Agglutinating

activity

Sugar Agglutinating

activity

Sugar Agglutinatin

g activity

Glucose - Ovalbumie - Glucosamine

HCl

-

Mannitol - Rhamnose - Saccharose -

Fructose - Maltose - Mucine ND

Galactose - D-Sorbitol - Arabinose -

Xylitol - Fetuin + Fucopyranno

side

-

Fucose - Inuline - Mannose -

-: presence of agglutinating activity, +: inhibition of the agglutinating activity, ND: No defined.


37

b. Inhibition limit of Fetuin

The minimuim concentration of fetuin causes inhibition of agglutination of lectins

from Moringa oleifera, is shown in the following table:

Table 16: Inhibition limit agglutination of fetuin with extracts M.oleifera seeds.

1/2

1/4

1/8

1/16

1/32

1/64

1/128

1/256

1/51

2

1/10

24

1/20

48

1/40

96

Fetuin +++ +++ ++ + - - - - - - - -

+: inhibition of agglutinating activity, - : presence of the agglutinating activity

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

Fetuin

Control test

Photo 12: Inhibition limit test of fetuin with M.oleifera.

The inhibition limit test of fetuin showed that it inhibits the hemagglutinating

activity of the lectin up to the

The inhition of agglutination by fetuin shows the similarity between the lectin in

this study and the Moringa lectin (MoL) isolated by Katre et al, 2008.

The non-inhibition of agglutination by fructose differeciates the lectin in this

study from the Water-soluble Moringa lectin which is specific to fructose. (Ferreira et

al., 2011).

Titer

value

Fraction

Titer

value

Sugar


38

c. Effect of temperature

The stability of the lectins with heat was investigated by subjecting it to different

temperatures for a period of 30 minutes. Heat treatment of the lectin in this study

revealed that it is a thermo stable protein.

High temperature is a powerful denaturating agent leading to protein unfolding

through breaking of hydrogen bonds that maintain protein structure. (Daggett and

Levitt., 1992).

Thermostability is a common feature of protein from MO seeds. MOCP isolated

by (Gassenschmidt et al., 1995) remained active after 5h heat treatment at 95°C.

Table 17: Effect of temperature on the agglutination test of Moringa oleifera.

T°C 30 40 50 60 70 80 90 100

HU +++ +++ ++ + ++ + + +

+++: very high agglutinating activity, ++: high agglutinating activity, +: low agglutinating activity,

HU: hemagglutinating unit.

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

30

40

50

60

70

Titer

value

T°C


39

80

90

100

Control test

Photo 13: Effect of temperature on the agglutination activity of Moringa oleifera.

The highest agglutinating activity of the lectin is observed at 30 and 40°C, and

an increase in the temperature relatively decreased the activity, but didn’t completely

eliminate it. No significant agglutinating activity difference was observed at

temperatures higher than 40°C.

Thermal stability of the lectin isolate indicates that the extraction of lectin from

Moringa Oleifera seeds can be done at room temperature.

Figure 11: Effect of temperature on the agglutinating activity.

From the figure above, we can deduce that our Moringa oleifera seeds contain

two lectins which have a high activity at 40°C and 70°C as shown by the peaks in the

curve.


40

d. Effect of pH on the agglutinating activity

The lectin shows remarkable pH stability, its activity being unaffected throughout

the entire range of pH from 3 to 10.This is in contrast to lectin from Parkia javanica

beans which is stable in pH 7–10. (Utarabhand and Akkayanont., 1995).

Table 18: Effect of pH on the agglutination test of Moringa oleifera.

PH 2 3 4 5 6 8 10

HU + +++ + ++ +++ + +

+++: very high agglutinating activity, ++: high agglutinating activity, +: low agglutinating activity,

HU: hemagglutinating unit.

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

2

3

4

5

6

8

10

Photo 14: Effect of pH on the agglutination test of Moringa oleifera.

Titer

value

PH


41

Figure 12: Effect of pH on the agglutinating activity.

From the figure above, we can deduce that the lectin activity is stable and has two

isoforms at pH 3 and 6.

e. Agglutination test with the ABO blood system

The human blood types have different sugar moieties on the surface of the cell as

shown in (Figure 06). Agglutination occurs when the lectin interacts with these sugar

moieties.

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

A

B

O

Photo 15: Agglutination test with the ABO blood system.

Titer

value

BLOOD

GROUP


42

The agglutinating activity was observed in the 3 blood groups, but was

significant in blood groups B and O, which showed an activity up to the 1/32 titer, while

blood group A showed a weak agglutinating activity up to the ¼ titer well.

The lectin is therefore considered to be non-blood group type specific. This is

similar to cMoL isolated by Santos et al., 2009 and the Crataeva tapia bark lectin

isolated by Araujo et al., 2012.

Non-blood type specificity of the lectin may be due to the presence of multiple

binding sites where it can recognise all the determinants for each blood type. (Aragones

and Merca., 1998).

f. Effect of EDTA on the agglutinating activity

The addition of EDTA to the lectin extract showed no significant difference in its

agglutinating activity despite the concentration of the EDTA.

⅟₂ ⅟₄ ⅛ ⅟₁₆ ⅟₃₂ ⅟₆₄ ⅟₁₂₈ ⅟₂₅₆ ⅟₅₁₂ ⅟₁₀₂₄ ⅟₂₀₄₈ ⅟₄₀₉₆

0

20

30

40

Titer

value

EDTA

(mM)


43

50

Control test

Photo 16: Effect of EDTA on the agglutinating activity.

The activity of the lectin decreased by a factor of 4 on addition of EDTA at any

concentration. The positive control test ( 0 EDTA) showed an agglutinating activity up

to the 1/512 titer value, while on addition of EDTA, at all concentrations (20, 30,

40,50), the agglutinating activity stopped at 1/128.

From these results, we can deduce that the lectin is not a metaloprotein, hence it

doesnot need metals for its activity.

Conclusion and

perspective

Conclusion and perspective

44

The key for efficient detection treatment and healing of pathological condition is

the biorecognition event.

Lectins have become a very popular class of biorecognition molecules due to their

ability to recognize carbohydrates and glycoconjugates in cells, tissues sections, and

biological fluids. They are isolated from distinct sources such as virus, bacteria, fungi,

algae, animals, and plants. (Luana et al., 2017).

In this study we extracted, isolated, purified and characterized phytolectins from

mature Moringa oleifera seeds. Extraction with PBS solution isolated two lectins from

the whole seed powder. Salting-out of the crude extract with ammonium sulfate showed

maximum precipitation at 0-50% (NH4)₂SO4 saturation and resulted in a 37.433

purification fold. Further purification was achieved by gel filtration on Sephadex G-50

column registering a 111.931 purification fold and 50% yield.

All fractions (crude extract, 0-50%, and chromatogratography) showed a positive

result of agglutinating activity with glutaldehyde-treated rabbit erythrocytes.

Furthermore, the ability to agglutinate all human blood types indicates that the lectin is

a non blood type specific.

When subjected to the inhibition test; the 0-50% (NH4)₂SO4 precipitate fraction

showed specificity to fetuin.

The two lectins were found to be thermostable, with optimal temperatures of 40

and 70°C. They were more active in a pH range of 3-6 where it exhibited maximum

agglutination.

The lectins can be considered as non metalloproteins since they can agglutinate

rabbit erythrocytes in presence of EDTA, a chelating agent.

Even though the study of lectins in Moringa oleifera seeds has already been

investigated, the lectins isolated in this study exhibit properties that are not particular to

the lectins in literature.

Perspectives;

This study stimulates more investigation on the following aspects on the lectins

from Moringa oleifera seeds,

Purification of the lectins by affinity chromatography.

Determination of the molecular weight of the lectins by SDS-PAGE.

Conclusion and perspective

45

The role of Moringa oleifera seed lectins in the purification of water.

Anti-microbiological roles of the lectins.

Immunomodulatory and anti-cancer activity tests of the lectins.

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Annex

Annex 01: Preparation of Buffer

Preparation of buffer (phosphate DI _ SODIQUE = PBS).

Annex 02: Sample Preparation in iron Exchange Chromatography.

Annex 03:

SHA (Specific Hemagglutinating Activity) = Total activity / Total protein

Purification fold = SHA of purified fraction / SHA of crude extract.

Yield= Original total activity that is found present in the purified fraction. Total

activity of purified fraction / Total activity of crude extract.

Products Quantity

Na2HPO4 1,44g

NaHPO4 0,28g

Kcl 0,2g

NaCl 8g

DISTILED WATER 1L

Annex

Annex 04: Preparation of Sugar

Preparation of sugar 400mM.

Sugar/glycoprotéins PBS(buffer)

400 mM 2 ml

Annex 05: Eye observation of color change of Bradford’s solution in

presence of lectins

Annex 06: Agglutination Test after Anion Exchange Chromatography

F01 F33 F45 Control

In presence

of the

protein;

lectin

Without

the

protein

BENSEGHIR Chaima NAMULODI Phiona Academic year: 2017-2018

Extraction, Isolation and Characterization of

LectinS from Moringa oleifera seeds

Abstract

The purpose of the research was to study the isolation, purification and

characterization of lectins from mature Moringa oleifera seeds. The lectins were extracted

from the seeds with phosphate-buffered saline (PBS) at pH 7.4, and partial purification

was accomplished by ammonium sulfate precipitation and gel filtration using Sephadex G-

50.

Agglutination using glutaraldehyde-treated rabbit erythrocytes and human blood types A,

B, O was carried to determine the presence and specificity of the lectin. Subsequently, the

Moringa oleifera seed lectins were classified as a complete non-specific lectin since it

agglutinated all human blood types and glutaldehyde-treated rabbit erythrocytes.

The results of carbohydrate specificity showed that the lectins had complex sugar

specificity to fetuin. The lectins were found to be thermally stable, and stable within the

pH range of 3-6. It was also considered as a non metallolectin since its activity was not

significantly affected by EDTA.

Keywords: Lectins, Extraction, Agglutination, Characterization, Moringa oleifera, Inhibition test.

Examination Board:

President: Dr. T. NOUADRI M. C. A. University of MENTOURI Brothers Constantine 1

Supervisor: Prof.Y. NECIB Pr. University of MENTOURI Brothers Constantine

Examiner: Dr. F. MEROUANE M. C. B. National School of Biotechnology

Date of presentation: 28 JUNE 2018

extraction, isolation and characterization of lectins from

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